Mycobacterium tuberculosis continues to exert a major influence on the health of a considerable proportion of the human population and despite the extraordinary technical advances made in the field in recent years we still struggle with putting this technology to effective use in unraveling the events key to the successful establishment of an intracellular infection. In this current application we propose to treat the infection process as a series of sequential environmental shifts or cues that induce specific programmed responses from the bacterium. The project proposes combining our established cell biological expertise in the manipulation and assessment of the intraphagosomal environment with our newly acquired expertise in microarray analysis and detection of bacterial promoter activity. In addition we propose to extend our experimental system from the macrophage to a lung model system employing alveolar macrophages, alveolar type I/II cells and lung organ culture. The specific aims of this proposal are as follows: 1. Detection and functional validation of the transcriptional changes in M. tuberculosis during infection of macrophages. This specific aim proposes to determine the functional significance of changes in bacterial gene transcription at defined stages during the infection process through the exploitation of microarray technology and recombinase in vivo expression technology. The infection process itself will be broken down into incremental steps, i.e. adherence, entry, survival in the early endosome and passage to the late endosome, through the use of pharmacological manipulation of the host cell, or bacterial mutants with altered intracellular trafficking behavior. Preliminary experiments demonstrate the power and validity of these approaches in identification of transcriptional changes. These changes will be validated further through determination of gene function and will be placed in the context of ongoing analyses into the environmental conditions existing within the endosomal continuum. 2. Functional analysis of these transcriptional profiles in an in vitro pulmonary model system. This specific aim proposes analysis of bacterial and host transcriptional profile changes within the context of a mixed cell culture system with alveolar cells, tracheal epithelial cells or lung organ culture. Both bacterial and host transcriptional changes will be monitored and dissected experimentally to determine how the environmental factors within the lung influence both bacterial and "host" cell behavior in these critical early stages of infection.